Alginate coating of Xenopus laevis embryos

Xenopus laevis eggs were coated, immediately after squeeze-stripping and fertilization, with a thin layer ( approximately 50 microm) of film based on one of three different types of alginates which varied in their mannuronic/guluronic acid ratio. The alginate was cross-linked with either Ca or Ba ions at three different concentrations. The developmental, survival, and hatching of these embryos and the swelling of their natural jelly coats or hydrocolloid coatings were studied over 7 days, while embryos were maintained in flowing aerated water at a ratio of 85 mL per embryo or at a very diminished ratio of 1.6 mL of sterile or nonsterile MMR solution per embryo. All experiments were conducted in triplicate at 20+/-1 degrees C. Oxygen was monitored continuously. Mineral content was determined in the alginate-jelly coat and within the embryos over time. The coating conferred major advantages when the ratio between the embryos and the surrounding medium was at a minimum under nonsterile conditions, perhaps as a result of the film's resistance to diffusion. In the studied systems, the coating seemed to postpone embryo hatching to a more developed stage. In addition, the coating served as a barrier to microbial contamination and thus improved survival prospects.     

Influence of Polymer Seed Coatings, Soil Raking, and Time of Sowing on Seedling Performance in Post-Mining Restoration

This study represents part of a broader investigation into novel seed broadcasting methodologies as a means to optimize rehabilitation techniques following sand mining. Specifically, the study investigated the use of polymer seed coatings, time of sowing application, and in situ raking of the topsoil to optimize seedling recruitment to site. For polymer seed coatings, an ex situ trial was undertaken to evaluate seed coating effects on seedling emergence. Results demonstrated that seed coatings did not significantly inhibit maximum emergence percentage of 10 Banksia woodland species (out of 11 evaluated), but coated seeds from four species were on average 2–6 days slower to emerge than noncoated seeds. Seed coatings were found to have a greater effect in situ, with more coated seeds emerging than noncoated seeds. Topsoil raking (following seed sowing) and time of sowing were found to have the greatest impact on seedling emergence, with higher emergence following topsoil raking (5‐ to 90‐fold increase) and sowing in May (late autumn) (1.4‐ to 12‐fold increase) rather than in July (mid‐winter). The implications for mining rehabilitation are discussed, and areas for further research are considered.     

Influence of collagen and chondroitin sulfate (CS) coatings on poly-(lactide-co-glycolide) (PLGA) on MG 63 osteoblast-like cells

Poly-(lactide-co-glycolide) (PLGA) is an FDA-approved biodegradable polymer which has been widely used as a scaffold for tissue engineering applications. Collagen has been used as a coating material for bone contact materials, but relatively little interest has focused on biomimetic coating of PLGA with extracellular matrix components such as collagen and the glycosaminoglycan chondroitin sulfate (CS). In this study, PLGA films were coated with collagen type I or collagen I with CS (collagen I/CS) to investigate the effect of CS on the behaviour of the osteoblastic cell line MG 63. Collagen I/CS coatings promoted a significant increase in cell number after 3 days (in comparison to PLGA) and after 7 days (in comparison to PLGA and collagen-coated PLGA). No influence of collagen I or collagen I/CS coatings on the spreading area after 1 day of culture was observed. However, the cells on collagen I/CS formed numerous filopodia and displayed well developed vinculin-containing focal adhesion plaques. Moreover, these cells contained a significantly higher concentration of osteocalcin, measured per mg of protein, than the cells on the pure collagen coating. Thus, it can be concluded that collagen I/CS coatings promote MG 63 cell proliferation, improve cell adhesion and enhance osteogenic cell differentiation.     

Investigation of the bioactivity and biocompatibility of different glass interfaces with hydroxyapatite, fluorohydroxyapatite and 58S bioactive glass

The current review investigates the bioactivity of different glass interfaces created on thin glass cover slips as substrates. The interfaces studied are plain glass, functionalized glass using 0.5 M and 5 M of sodium hydroxide (NaOH) for 24 hrs, and glass coated with bioactive 58S Bioglass (58S). A biomimetic method, involving the exposure of the three interfaces to 1.5 times simulated body fluid (SBF) tests the bioactivity of the interfaces via creation of layer of Hydroxyapatite (HA). Fluorinated SBF will precipitate fluorine doped HA (FHA) on a bioactive interface. Higher concentration of 1.5 times of SBF used in this study intended to accelerate the formation of HA and FHA layer over the substrate. HA and FHA is found to be precipitated on the thinly coated 58S. This paper, study also the thin film coatings of three forms of bioceramics - bioactive 58S, HA and FHA. The study, also proposes to draw a relation between the morphology of HA particles with duration of exposure to SBF, the effects of fluorine on the morphology and the cell interaction with bioactive 58S, HA and FHA interfaces using pre-differentiated osteoblastic MC3T3 cells. The analysis of cells in this study is confined to three parameters that include the attachment, proliferation and viability of cells. Tests employed for the analysis of the thin film coating of HA and FHA is restricted to qualitative X-Ray Diffraction and quantitative Field Emission Scanning Electron Microscope. Other mechanical tests such as shear test are not used to test the mechanical properties of this thin layer, due to the fact that the thin film is too thin for such analysis.     

Drying Dynamics of Solution‐Processed Perovskite Thin‐Film Photovoltaics: In Situ Characterization,Modeling, and Process Control

A key challenge for the commercialization of perovskite photovoltaics is the transfer of high‐quality spin coated perovskite thin‐films toward applying industry‐scale thin‐film deposition techniques, such as slot‐die coating, spray coating, screen printing, or inkjet printing. Due to the complexity of the formation of polycrystalline perovskite thin‐films from the precursor solution, efficient strategies for process transfer require advancing the understanding of the involved dynamic processes. This work investigates the fundamental interrelation between the drying dynamics of the precursor solution thin‐film and the quality of the blade coated polycrystalline perovskite thin‐films. Precisely defined drying conditions are established using a temperature‐stabilized drying channel purged with a laminar flow of dry air. The dedicated channel is equipped with laser reflectometry at multiple probing positions, allowing for in situ monitoring of the perovskite solution thin‐film thickness during the drying process. Based on the drying dynamics as measured at varying drying parameters, namely at varying temperature and laminar air flow velocity, a quantitative model on the drying of perovskite thin‐films is derived. This model enables process transfer to industry‐scale deposition systems beyond brute force optimization. Via this approach, homogeneous and pinhole‐free blade coated perovskite thin‐films are fabricated, demonstrating high power conversion efficiencies of up to 19.5% (17.3% stabilized) in perovskite solar cells.     

Coating of coverslips with glow-discharged carbon promotes cell attachment and spreading probably due to carboxylic groups

BACKGROUND: For high-resolution microscopy, cells have to be analyzed through thin glass coverslips. Therefore, it is necessary to culture cells on coverslips for preservation of cell morphology. We found cell attachment and spreading to be relatively slow processes, even when cells were plated on coated coverslips. This slowness presents a problem, particularly when synchronized cell populations are used. METHODS: In this paper, we present a method that is based on glow-discharged carbon coating of coverslips which promotes rapid attachment and spreading of cells, enabling rapid analysis of cells after plating. Results obtained with carbon-coated coverslips were compared with those of other types of coating. Two fibroblast lines, an epithelial cell line, and a carcinoma cell line were tested. RESULTS AND CONCLUSIONS: All cell lines showed a rapid adhesion on carbon-coated coverslips. With fibroblasts we found the carbon coating to be superior to other coatings tested, mainly because the carbon did not influence cell morphology. Using synchronized or irradiated cells produced similar results. The superior performance of carbon coating is probably due to carboxylic groups on the glow-discharged carbon layer. The carbon layer does not interfere with microscopy or immunocytochemical staining procedures.     

Antibacterial activity of plastics coated with silver-doped organic-inorganic hybrid coatings prepared by sol-gel processes

Silver-doped organic-inorganic hybrid coatings were prepared starting from tetraethoxysilane- and triethoxysilane-terminated poly(ethylene glycol)-block-polyethylene by the sol-gel process. They were applied as a thin layer (0.6-1.1 microm) to polyethylene (PE) and poly(vinyl chloride) (PVC) films and the antibacterial activity of the coated films was tested against Gram-negative (Escherichia coli ATCC 25922) and Gram-positive (Staphylococcus aureus ATCC 6538) bacteria. The effect of several factors (such as organic-inorganic ratio, type of catalyst, time of post-curing, silver ion concentration, etc.) was investigated. Measurements at different contact times showed a rapid decrease of the viable count for both tested strains. The highest antibacterial activity [more than 6 log reduction within 6 h starting from 106 colony-forming units (cfu) mL-1] was obtained for samples with an organic-inorganic weight ratio of 80:20 and 5 wt % silver salt with respect to the coating. For the coatings prepared by an acid-catalyzed process, a high level of permanence of the antibacterial activity of the coated films was demonstrated by repeatedly washing the samples in warm water or by immersion in physiological saline solution at 37 degrees C for 3 days. The release of silver ions per square meter of coating is very similar to that previously observed for polyamides filled with metallic silver nanoparticles; however, when compared on the basis of Ag content, the concentration of silver ions released from the coating is much higher than that released from 1 mm thick specimens of polyamide (PA) filled with silver nanoparticles. Transparency and good adhesion of the coating to PE and PVC plastic substrates without any previous surface treatment are further interesting features.     

Removable colored coatings based on calcium alginate hydrogels

This article describes the creation of a nontoxic, biodegradable coating using calcium alginate and FD&C approved dyes. The coating is robust but is rapidly removed upon treatment with disodium ethylenediamine tetraacetate (EDTA). Dye leaching from calcium alginate films was studied, and it was determined that the efficiency of dye retention is proportional to the degree of cross-linking. Degradation rates were studied on calcium alginate beads serving as a model for a coating. We determined that degradation rates depend on the gel's cross-linking and on the amount of EDTA used. Bead size also influenced the degradation rates; smaller beads degraded faster than larger beads. We show that the coating can be used as an easily removable and environmentally friendly logotype on an artificial turf surface. Applications of these coatings can be extended to food, cosmetic, medicinal, and textile uses and to wherever nontoxic, easily removable colored coating is desired.     

Layer-by-layer Synthesis and Transfer of Freestanding Conjugated Microporous Polymer Nanomembranes

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in combination with their outstanding thermal and chemical stability, and low densities. However, their insoluble nature causes problems in their processing since usually applied techniques such as spin coating are not available. Especially for membrane applications, where the processing of CMP as thin films is desirable, the processing problems have hindered their commercial application.Here we describe the interfacial synthesis of CMP thin films on functionalized substrates via molecular layer-by-layer (l-b-l) synthesis. This process allows the preparation of films with desired thickness and composition and even desired composition gradients.The use of sacrificial supports allows the preparation of freestanding membranes by dissolution of the support after the synthesis. To handle such ultra-thin freestanding membranes the protection with sacrificial coatings showed great promise, to avoid rupture of the nanomembranes. To transfer the nanomembranes to the desired substrate, the coated membranes are upfloated at the air-liquid interface and then transferred via dip coating.     

The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations

The transfer of molecules from the cell surface to the early endosomes is mediated by preendosomal vesicles. These vesicles, which have pinched off completely from the plasma membrane but not yet fused with endosomes, form the earliest compartment along the endocytic route. Using a new assay to distinguish between free and cell surface connected vesicle profiles, we have characterized the preedosomal compartment ultrastructurally. Our basic experimental setup was labeling of the entire cell surface at 4 degrees C with Con A-gold, warming of the cells to 37 degrees C to allow endocytosis, followed by replacing incubation medium with fixative, all within either 30 or 60 s. Then the fixed cells were incubated with anti-Con A-HRP to distinguish truly free (gold labeled) endocytic vesicles from surface-connected structures. Finally, analysis of thin (20-30 nm) serial sections and quantification of vesicle diameters were carried out. Based on this approach it is shown that the preendosomal compartment comprises both clathrin-coated and non-coated endocytic vesicles with approximately the same frequency but with distinct diameter distributions, the average noncoated vesicle being smaller (95 nm) than the average coated one (110 nm). In parallel experiments, using an anti-transferrin receptor gold-conjugate as a specific marker for clathrin-dependent endocytosis it is also shown that uncoating of coated vesicles plays only a minor role for the total frequency of noncoated vesicles. Furthermore, after perturbation of clathrin-dependent endocytosis by potassium depletion where uptake of transferrin is blocked, noncoated endocytic vesicles with Con A-gold, but not coated vesicles, exist already after 30 and 60 s. Finally, it is shown that the existence of small, free vesicles in the short-time experiments cannot be ascribed to recycling from the early endosomes.     

Studies of the mechanical properties and the fermentation behavior of double layer alginate–chitosan beads,using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> entrapped cells

Double layer alginate beads coated with chitosan were constructed for the entrapment of yeast cells used in alcoholic fermentations. Several construction parameters of the beads were studied. Among these parameters were the composition of the inner and the outer layer, the initial cell loading, the concentration of chitosan in the coating solution. Improved bead behavior was noticed by the use of chitosan as a coating agent to double layer alginate beads. The mechanical strength and the stability of the beads were enhanced. The polyelectrolyte complex membrane of alginate–chitosan reduced significantly the leakage of the entrapped cells into the medium. The aim of this work was to define the optimal conditions for the construction of the double layer alginate beads coated with chitosan with the purpose of using them for the fermentation of carbohydrates. This paper is based on a presentation at the “International Congress on Bioprocesses in Food Industries – ICBF 2006” conference, Patras 2006     

Spectrophotometric quantification of lactic bacteria in alginate and control of cell release with chitosan coating

Lactococcus lactis ssp. cremoris was entrapped within a Ca-alginate matrix, and an in situ spectrophotometric method for monitoring cell population in calcium alginate beads described. The intracapsular cell population can be estimated by measuring the optical density of beads containing cells, using cell-free beads as reference, or by measuring absorbance of a liquified bead suspension. Alginate beads, and beads coated with chitosan type I, II, and I and II mixtures, were examined for cell release. Lower viscosity chitosan (type I) coatings reduced cell release by a factor of 100 from10⁵ cfu ml⁻¹ to 10³ cfu ml⁻¹ after 6 h of fermentation. Reuse of chitosan I coated alginate beads also showed a reduction in cell release by a factor of 100. Cell loading and initial cell growth within the beads greatly affected cell release. Reducing the initial cell release would lower the overall levels of cell release throughout the fermentation. Compared to non-immobilized cultures, a 20–40% reduction in the lactic acid production rate was observed for alginate beads and chitosan I coated alginate beads, respectively. This reduction can be compensated for by increasing the intracapsular cell loading during immobilization, or before the onset of fermentation.     

The role of conditioning film formation in Pseudomonas aeruginosa PAO1 adhesion to inert surfaces in aquatic environments

Bacterial initial adhesion to inert surfaces in aquatic environments is highly dependent on the surface properties of the substratum, which can be altered significantly by the formation of conditioning films. In this study, the impact of conditioning films formed with extracellular polymeric substances (EPS) on bacterial adhesion was investigated. Adhesion of wild type Pseudomonas aeruginosa PAO1 to slides coated with model EPS components (alginate, humic substances, and bovine serum albumin (BSA)) was examined. Surface roughness of conditioning film coated slides was evaluated by atomic force microscopy (AFM), and its effect on the bacterial initial adhesion was not significant. X-ray photoelectron spectroscopy (XPS) studies were performed to determine the elemental surface compositions of bacterial cells and substrates. Results showed that bacterial adhesion to bare slides and slides coated with alginate and humic substances increased as ionic strength increased. Conversely, BSA coating enhanced bacterial adhesion at low ionic strength but hindered adhesion at higher ionic strength. It was concluded that forces other than hydrophobic and electrostatic interactions were involved in controlling bacterial adhesion to BSA coated surfaces. A steric model for polymer brushes that considers the combined influence of steric effects and DLVO interaction forces was shown to adequately describe the observed bacterial adhesion behaviors.     

Emulsion strategies in the microencapsulation of cells: pathways to thin coherent membranes

Microencapsulation of cell spheroids in an immunoselective, highly biocompatible, biomembrane offers a way to create viable implantation options in the treatment of insulin-dependent diabetes mellitus (IDDM). Traditionally the encapsulation process has been achieved through the injection/extrusion of alginate/cell mixtures into a calcium chloride solution to produce calcium alginate capsules around the cells. A novel alternative is explored here through a procedure using an emulsion process to produce thin adherent calcium alginate membranes around cell spheroids. In this study, a thorough investigation has been used to establish the emulsion process parameters that are critical to the formation of a coherent alginate coat both on a model spheroid system and subsequently on cell spheroids. Optical and fluorescence microscopy are used to assess the morphology and coherence of the calcium alginate/poly-L-ornithine/alginate (APA) capsules produced.     

Enzymic thin film coatings for bioactive materials

Bioactive materials have been explored for a broad range of applications including biocatalysts, biosensors, antifouling membranes and other functional and smart materials. We report herein a unique method for preparation of bioactive materials through a spin coating process. Specifically, we investigated the preparation of protease Subtilisin Carlsberg-coated plastic films and examined their activities for hydrolysis of chicken egg albumin (CEA). The process generated enzymic coatings with a typical loading of 13 microg/cm2, retaining 46% of the enzyme activity for hydrolysis of CEA in aqueous solutions. Interestingly, the surface-coated protease thin film not only catalyzed the hydrolysis of CEA in aqueous solutions, but also showed good activity for solid-state CEA that was coated on top of the enzyme thin film.     

Composition Engineering in Doctor‐Blading of Perovskite Solar Cells

Organic–inorganic halide perovskite (OIHP) solar cells with efficiency over 18% power conversion efficiency (PCE) have been widely achieved with lab scale spin‐coating method which is however not scalable for the fabrication of large area solar panels. The PCEs of OIHP solar cells made by scalable deposition methods, such as doctor‐blading or slot‐die coating, have been lagging far behind than spin‐coated devices. In this study the authors report composition engineering in doctor‐bladed OIHP solar cells with p–i–n planar heterojunction structure to enhance their efficiency. Phase purer OIHP thin films are obtained by incorporating a small amount of cesium (Cs⁺) and bromine (Br^?) ions into perovskite precursor solution, which also reduces the required film formation temperature. Pinhole free OIHP thin films with micrometer‐sized grains have been obtained assisted by a secondary grain growth with added methylammonium chloride into the precursor solution. The OIHP solar cells using these bladed thin films achieved PCEs over 19.0%, with the best stabilized PCE reaching 19.3%. This represents a significant step toward scalable manufacture of OIHP solar cells.     

Simplified construction and characterization of yeast artificial chromosome libraries.

Three yeast artificial chromosome (YAC) libraries were constructed using two human cell lines and the pYAC-RC vector. The main differences from the previously described methods were: i) genomic DNA was digested in low melting point (LMP) agarose blocks with the rare cutting enzyme ClaI; ii) DNA was ligated in melted LMP agarose after agarase treatment; iii) spheroplast regeneration plating was done in calcium alginate thin layer. In addition, a panel of PCR primers was used to identify quickly the presence in the libraries of repetitive and single copy human DNA sequences.     

Modeling of spherical fluorescent glucose microsensor systems: design of enzymatic smart tattoos

A two-substrate mathematical model of microspherical optical enzymatic glucose sensors is presented. The sensors are based on the well-known oxidation of glucose by glucose oxidase, and are constructed by the encapsulation of glucose oxidase within hydrogel microspheres coated with ultrathin polyelectrolyte multilayer films. In order to measure glucose via changes in oxygen concentration, a fluorescent oxygen indicator is co-encapsulated with the enzyme. The model was used to predict the temporal and spatial distributions of glucose and oxygen within the sphere for step increases in bulk glucose concentration. In addition, the model was used to observe the effect of varying sensor parameters, namely sphere size, film thickness, enzyme concentration, and mass transport of substrate and co-substrate within the sphere and film coatings, on the response of the sensors. A major finding was that the application of {PSS/PAH} films as thin as 12 nm can drastically improve the sensor performance over uncoated sensors based on calcium alginate microspheres. The model is proposed as an important tool for a priori design of these complex sensor structures.     

Induction of the Epstein-Barr virus latent membrane protein 2 antigen-specific cytotoxic T lymphocytes using human leukocyte antigen tetramer-based artificial antigen-presenting cells

Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membraneprotein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies,such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma.However,the therapeutic amount ofCTLs is often hampered by the limited supply of antigen-presenting cells.To address this issue,an artificialantigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetramericcomplex,anti-CD28 antibody and CD54 molecule to a cell-sized latex bead,which provided the dual signalsrequired for T cell activation.By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral bloodmononuclear cells from HLA-A2 positive healthy donors,LMP2 antigen-specific CTLs were induced andexpanded in vitro.The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell,the cytotoxicity was inhibited bythe anti-HLA class Ⅰ antibody (W6/32).These results showed that LMP2 antigen-specific CTLs could beinduced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC.Thus,aAPCs coated with an HLA-pLMP2 complex,anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specificCTLs for adoptive immunotherapy.     

Ultrastructural analysis of the organization and distribution of insulin receptors on the surface of 3T3-L1 adipocytes: rapid microaggregation and migration of occupied receptors

Monomeric ferritin-insulin and high-resolution electron microscopic analysis were used to study the organization, distribution, and movement of insulin receptors on differentiated 3T3-L1 adipocytes. Analysis of the binding to prefixed cells showed that insulin initially occupied single and paired receptors preferentially located on microvilli. The majority of receptors (60%) were found as single molecules and 30% were pairs. In 1 min at 37% C, 50% of the receptors on nonfixed cells were found on the intervillous plasma membrane and more than 70% of the total receptors had microaggregated. By 30 min only 7% of the receptors were single or paired molecules on microvilli. The majority were on the intervillous membrane, with 95% of those receptors in groups. The receptor groups on the intervillous plasma membrane could be found in both noncoated invaginations and coated pits. The concentration of occupied receptors in the noncoated invaginations and the coated pits was similar; however, ten times more noncoated invaginations than coated pits contained occupied insulin receptors. The observations in this study contrast with those reported on rat adipocytes using identical techniques (Jarett and Smith, 1977). Insulin receptors on adipocytes were initially grouped and randomly distributed over the entire cell surface and did not microaggregate into larger groups. Insulin receptors on rat adipocytes were found in noncoated invaginations but were excluded from the coated pits. The differences in the organization and behavior of the insulin receptor between rat and 3T3-L1 adipocytes suggest that the mechanisms regulating the initial organization of insulin receptors and the aggregation of occupied receptors may be controlled by tissue-specific processes. Since both of these cell types are equally insulin sensitive, the differences in the initial organization and distribution of the insulin receptors on the cell surface may not be related to the sensitivity or biological responsiveness of these cells to insulin but may affect other processes such as receptor regulation and internalization. On the other hand, the microaggregates of occupied receptors on both cell types may relate to biological responsiveness.